Protocol for the in vitro reconstruction of site-specifically phosphorylated RNA Pol II to identify the recruitment of novel transcription regulators

Summary The repetitive C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) becomes differentially phosphorylated throughout the transcription cycle. Here, we present a protocol to site-specifically phosphorylate the CTD of RNAPII by leveraging the specificity of well-characterized CTD kinases. We describe the steps for optimal phosphorylation of the CTD and the preparation of nuclear protein extract. This protocol can be used to identify the interactome of a phospho-CTD and has the potential to identify novel RNAPII-binding proteins. For complete details on the use and execution of this protocol, please refer to Moreno et al.1


Highlights
Purify the unphosphorylated C-terminal domain (CTD) of RNA polymerase II Site-specifically phosphorylate residues from CTD heptad repeats Identify the interactome of a CTD with specific phosphorylation sites Many groups have sought to characterize RNAPII protein-protein interactions, and as a result a myriad of different approaches have emerged to identify RNAPII interactomes at different stages of transcription. 4Affinity purification mass spectrometry (AP-MS) is one such method that has been leveraged to identify RNAPII-binding proteins.Traditionally, this strategy uses an antibody against a suspected RNAPII-binding protein to do the pull-down experiment.If the suspected RNAPII-binding protein does associate with the transcription complex, subunits of RNAPII should be top hits in the pull-down results.][7][8][9][10] Another strategy employed to characterize the RNAPII interactome was to immunopreciptate the RNAPII complex from cell lysate and then subsequently immunopreciptate out different RNAPII complexes using antibodies that are specific to different CTD phosphorylation signatures. 11,12This method has been used to characterize the interactomes of RNAPII complexes phosphorylated at either the Y1, S2, T4, S5, or S7 positions in yeast.However, the scope of this strategy is dominated by the recognition profile of phospho-specific CTD antibodies, which often struggle to recognize the CTD in a site-specific manner when neighboring residues are post translationally modified. 13,14Furthermore, since RNAPII is hyperphosphorylated during transcription, it will be difficult to discern which proteins are recruited by specific phosphorylation signatures.Another strategy that has been used with some success has been to mutate the CTD in some manner, disrupting site-specific phosphorylation, and use the mutant CTD in a pulldown experiment to better understand the role of different phosphorylation sites in recruiting proteins to the RNAPII complex during transcription. 157][18] Our strategy fits into this final category.In this protocol a GST-tagged yeast CTD, composed entirely of consensus repeats, is phosphorylated with kinases of known specificity.The phosphorylated CTD is subsequently used in a label-free proteomics experiment where the "bait" phospho-CTD is incubated with HEK293 nuclear lysate.The advantage of the strategy employed in this protocol is that we can directly link protein recruitment to a specific RNAPII post translational modification, since we are using a homogenously phosphorylated substrate.Furthermore, the hyperphosphorylated long CTD mimics the physiological state of the CTD during transcription and greatly increases the signal/noise ratio for a more confident identification of phospho-RNAPII interactomes.
0][21][22] Though we used HEK293 cells for our experiments and reconstructed Ser2 and Ser5 phosphorylation on the CTD in vitro, this protocol can be expanded to other phosphorylation sites, cell lines, or species.Additionally, this protocol can be adapted if other CTD kinases are characterized and can be used to identify the interactomes for different CTD constructs (i.e., distal vs. proximal CTD, or CTDs of varying lengths, etc.).Note: Ensure the final volume of trypsin/DMEM is 50 mL or less so that the entire solution fits into a 50 mL conical vial that can be placed into a centrifuge to pellet cells. .This is the volume that the pelleted cells take up in the tube.The PCV will be used as the volume for wash steps during protein extraction steps.

SOC media
Dissolve the following reagents in 40 mL of ddH 2 O: 1.00 g tryptone 0.25 g yeast extract 0.025 g NaCl Add 500 mL of 250 mM KCl to the solution and dilute the solution to 49 mL.Adjust the pH of the solution to 7.0 with 1 M NaOH.Autoclave the solution, and then let the solution cool to the touch.Add 250 mL of sterile 2 M MgCl 2 and 1 mL of sterile 1 M glucose.Aliquot 800 mL prepared SOC media into microcentrifuge tubes and freeze at À20 C for future use.SOC media can be stored at À20 C indefinitely.
CRITICAL: 2-Mercaptoethanol is a skin irritant and is toxic if swallowed or inhaled.Please handle with appropriate lab safety gear in a fume hood.

Timing: 5 days
This section gives step by step instructions for purifying a 26X CTD recombinant protein tagged with an N-terminal hexahistidine glutathione S-transferase (HGST) that will be used as the substrate for phosphorylation and subsequently used as bait in the pull-down experiment.
1. Transform the HGST-26X CTD pET-28a plasmid into E. coli BL21 (DE3) cells.a. Add 50 ng of the HGST-26X CTD plasmid to a 50 mL aliquot of E. coli BL21 (DE3) cells.b.Incubate plasmid-cell solution on ice for 30 min.c.Place tube containing the plasmid-cell solution in a water bath set to 42 C for 60 s to heat shock.d.Place tube on ice for 5 min after heat shock.e. Transfer heat shocked cells to a microcentrifuge tube containing 800 mL SOC media.f.Place tube in incubator set to 37 C and 200 rpm and shake sample for 1 h to recover cells.g.Spin cells down at 3,000 g for 3 min at 20 C-22 C. h.Discard supernatant and resuspend cells in 50 mL of LB media.i. Plate cells on LB agar plates that contain 50 mg/mL kanamycin.j.Place LB/Kanamycin plates in a 37 C incubator for a minimum of 16 h.Colonies should be visible after 24 h. 2. Express HGST 26X CTD.
a. Add 50 mL of 50 mg/mL kanamycin to Erlenmeyer flask that contains 50 mL of LB media, so that the final concentration of kanamycin in the LB media is 50 mg/mL.b.Inoculate the 50 mL LB/Kanamycin flask with cells from a colony on the plate from step 1j.c.Grow starter culture in a shaking incubator for 16-18 h at 37 C with shaking at 200 rpm.d.Inoculate two 1 L Luria-Bertani broth (LB) cultures with 10 mL starter culture and 50 mg/mL kanamycin.e. Grow 1 L cultures at 37 C with shaking at 200 rpm until the OD reaches 0.6.f.Induce cultures by adding IPTG so that the final IPTG concentration in each 1 L culture is 0.5 mM and reduce the temperature of the shaker to 16 C to promote proper protein folding during expression.g.Let cultures incubate in the shaker 16-18 h at 16 C and 200 rpm.e. Wash the column with 10 CV of CTD lysis buffer.f.Elute protein with 4 CV of CTD elution buffer.g.Run SDS-PAGE to confirm purity.An example SDS-PAGE for all HGST-26X CTD purification steps is shown in Figure 1A. Figure 1C shows HGST-26X CTD purity after size-exclusion chromatography.5. Further purify HGST-26X CTD by size-exclusion chromatography.
a. Concentrate eluted protein to 5 mL using a protein concentrator with a molecular weight cut off of 10 kDa.b.Buffer exchange eluted protein to CTD SEC buffer by adding 5 mL of SEC buffer to concentrator at a time and reconcentrating the sample to 5 mL.c.Repeat step 5b at least 4 additional times to ensure that a minimum of 25 mL of CTD SEC buffer is used to minimize the concentration of imidazole in solution before loading onto the column.d.Concentrate the buffer exchanged sample to 2 mL.e. Equilibrate Superdex 75 size-exclusion column (GE) with CTD SEC buffer.f.Run sample over SEC and pool fractions from peak corresponding to $49 kDa as shown in Figure 1B.g.Check protein concentration and concentrate to 4 mg/mL.

Purify DYRK1a kinase
Timing: 5 days This step is an example protocol for purifying an active CTD kinase that will be used to phosphorylate the CTD substrate for the pull-down experiment.a. Resuspend cells from both pellets in 50 mL DYRK1a lysis buffer.
Note: When using TB media, the cell pellets are typically larger than the cell pellets produced from LB media.Thus, you can use more lysis buffer to resuspend the cell pellet if needed.Since the His-DYRK1a will be initially purified by Ni-NTA affinity resin, the final volume of the cell lysate does not affect this purification step.
b. Sonicate resuspended cells with five 30 s cycles.For each 30 s cycle, set the on time to 1 s, the off time to 5 s, and the amplitude to 90 A. Let the sample rest 3 min on ice between cycles.
Note: Keep the sample in ice throughout the sonication process to prevent the sample from heating up during sonication.a. Concentrate eluted protein to 5 mL using a protein concentrator with a molecular weight cut off of 10 kDa.b.Buffer exchange eluted protein to DYRK1a SEC buffer by adding 5 mL of SEC buffer to concentrator at a time and reconcentrating the sample to 5 mL.c.Repeat step 10 b at least 4 additional times to ensure that a minimum of 25 mL of CTD SEC buffer is used to minimize the concentration of imidazole in solution before loading onto the column.d.Concentrate the buffer exchanged sample to 2 mL.e. Equilibrate Superdex 200 size-exclusion column (GE) with DYRK1a SEC buffer.f.Run sample over SEC and pool fractions from peak corresponding to $40 kDa.An example chromatogram is shown in Figure 2A.g.Check protein concentration and concentrate as needed.
Note: For this protocol, you do not need a super concentrated kinase sample.A concentration of 0.5 mg/mL is sufficient.

Purify ERK2 kinase
Timing: 5 days This step is an example protocol for purifying an active CTD kinase that will be used to phosphorylate the CTD substrate for the pull-down experiment.e. Transfer heat shocked cells to a microcentrifuge tube containing 800 mL SOC media.f.Spin cells down at 3,000 g for 3 min at 20 C-22 C. g.Discard supernatant and resuspend cells in 50 mL of LB media.h.Plate cells on LB agar plates that contain 100 mg/mL ampicillin.i. Place LB/Ampicillin plates in a 37 C incubator for a minimum of 16 h.Colonies should be visible after 24 h.12. Express ERK2/MEK1.a. Add 100 mL of 50 mg/mL ampicillin to Erlenmeyer flask that contains 50 mL of LB media, so that the final concentration of ampicillin in the LB media is 100 mg/mL b.Inoculate the 50 mL LB/Ampicillin flask with cells from a colony on the plate from step 11j.c.Grow starter culture in a shaking incubator 16-18 h at 37 C with shaking at 200 rpm.Note: ERK2 was co-expressed with MEK1 (R4F).ERK2 has an N-terminal hexahistidine tag, while MEK1 (R4F) does not, thus most of the MEK1 (R4F) should be separated from the desired ERK2 protein during the previous Ni-NTA affinity purification.However, as an additional polishing step we do anion exchange.Both ERK2 and MEK1 (R4F) are approximately 41.0 kDa, thus size-exclusion chromatography will not be useful in this instance.However, the predicted isoelectric point of ERK2 (pI = 6.50) is slightly less acidic than that of MEK1 (pI = 6.03), thus anion exchange chromatography can be used in this case to remove any trace amounts of MEK1 (R4F) that remain in the sample.d.Load dialyzed sample on Mono Q column and elute protein with a 50-1000 mM NaCl gradient using ERK2 Mono Q buffer B. e. Run SDS-PAGE gel to confirm purity as shown in Figure 3.A band for ERK2 should appear at $41 kDa.f.Pool fractions containing ERK2.Check protein concentration and concentrate as needed.
Note: For this protocol, you do not need a super concentrated kinase sample.Concentrating to 0.5 mg/mL is sufficient.

Prepare CTD substrate
Timing: 1 day This step details conditions to site-specifically phosphorylate the CTD substrate in vitro in preparation for a pull-down experiment.Note: You want a 1:1 ratio of GST bead volume to kinase reaction solution, thus if you plan to add 100 mL kinase reaction mix to the beads then you will want 200 mL of 50% beads.
i.You will need 1 tube of beads per sample.Use a fresh pipette tip to pipette beads into each tube to ensure you are as accurate as possible when pipetting the beads, since they are very sticky!b.Centrifuge beads at 4,000 g for 2 min at 20 C-22 C and remove the supernatant.
CRITICAL: Be careful not to disturb the beads.c.Wash the beads by adding 1 mL of buffer C to each tube and incubating the tubes on a nutator for 5 min at 4 C. i. Wash the beads a total of 3 times.Spin the beads down as done in step 17b after each wash.d.After all washes are complete carefully remove the supernatant from the beads.e. Add 100 mL of the phosphorylated HGST-26X CTD mix to the washed beads.The remaining 20 mL of the HGST-26X CTD can be stored at À20 C until mass spectrometry can be done to confirm phosphorylation.f.Increase the final volume of the solution in each tube to 1 mL by adding approximately 800 mL of buffer C. g.Bind HGST-26X CTD to the GST beads by incubating the tubes on a nutator 16

Prepare nuclear lysate
Timing: 1 day This section details steps to extract nuclear lysate from HEK293 cells to be used as the ''prey'' in a pull-down experiment.Since RNA polymerase II is a nuclear protein complex, we used nuclear lysate for our pull-down experiment to reduce the appearance of hits that are not biologically relevant.

Timing: 1-2 days
This step details how to set up the pull-down experiment with HEK293 nuclear lysate ''prey'' and the phosphorylated CTD ''bait.''21.Wash the GST beads after incubating with HGST-26X CTD.
a. Wash the GST beads by adding 1 mL of buffer C to each tube and incubating the tubes on a nutator for 5 min at 4 C. Wash the CTD-bound GST beads a total of 3 times.Spin the beads down as done in step 17b after each wash.b.After all washes are complete carefully remove the supernatant from the beads.b.Wash the beads by adding 1 mL of low salt buffer to each tube and incubate the tubes on a nutator for 5 min at 4 C. i. Wash the CTD-bound beads a total of 2 times with the low salt buffer.Spin the beads down as done in step 17b after each wash.c.Wash the beads by adding 1 mL of high salt buffer to each tube and incubate the tubes on a nutator for 5 min at 4 C.

Note:
We used two buffers with different ionic strengths to help reduce nonspecific binding interactions.
i. Wash the CTD-bound beads a total of 3 times with the high salt buffer.Spin the beads down as done in step 17b after each wash.d.Add 100 mL of elution buffer and incubate the tubes on a nutator for at least 2 h at 4 C. e. Centrifuge tubes at 4,000 g for 2 min at 4 C. f.Collect the supernatant as your pull-down samples for MS/MS analysis.
Note: Nuclear lysates can be stored at À20 C for up to a week.

EXPECTED OUTCOMES
After submitting samples for MS/MS analysis, you will obtain a table of proteins hits with corresponding peptide-spectrum match (PSM) counts for each hit in every sample as shown in Table 1.
From these data, the log2 fold change (FC) and Àlog10 p value can be calculated and plotted in a volcano plot as shown in Figure 5.

QUANTIFICATION AND STATISTICAL ANALYSIS
Eluted proteins from step 23f were prepared for tandem MS as stated in Moreno et al. 1 Briefly, proteins were separated using a 75 mM 3 25 cm Acclaim PepMap100 C-18 column (Thermo Scientific) and analyzed online by nanoelectrospray-ionization tandem MS on a Thermo Scientific Fusion Tribrid Orbitrap mass spectrometer.MS1 scans were collected at high resolution, and MS2 scans were acquired in the ion trap in rapid scan mode with fragmenting by collision-induced dissociation.Proteome Discoverer 2.3 (Thermo Scientific) was used to identify proteins by searching against the human reference proteome for UniProt.A false discovery rate of 1% was used to identify peptides, and a z-score was calculated to estimate the changes in protein abundance between control and kinase-treated samples according to Floyd et al. 23 Counts from the two control replicates and the two kinase-treated replicates were imputed to generate missing values.Then the data were quantile normalized and log2 transformed.p values were calculated using a two-tailed t-test, and significantly enriched proteins were defined as

LIMITATIONS
The use of kinases to phosphorylate the CTD of RNAPII in vitro relies on previous well-characterized kinases for their specificity and kinase activity.4,25 Thus, our ability to discern hits for different CTD post-translational modifications is limited to the phosphorylation patterns that we can recapitulate in vitro.Other strategies must be employed to characterize the interactomes of CTD PTMs, such as pThr4, until a kinase is identified that can place these CTD PTMs in vitro.Additionally, our strategy used a 26X WT CTD mostly of consensus sequence as the substrate for our pull-down experiment.However, the human 52X CTD might recruit additional factors since some heptads deviate from the consensus sequence.Interactomes for divergent sequences have yet to be studied but can be easily attained with our strategy.Finally, our experimental design leverages the proteomics experiment described in this protocol to identify lead RNAPII-binding proteins.The output of our approach yields a list of hundreds of lead proteins that must further be characterized via other methods, such as those shown in Moreno et al., to validate the hit as a RNAPII-binding protein. 1

TROUBLESHOOTING Problem 1
Contaminating skin, cytosolic, or mitochondrial proteins appear as hits in final result table.

Potential solution
No matter how careful you are when doing the nuclear extraction steps, contaminating proteins can appear in the raw output data.To mitigate this issue, we suggest using the ID mapping function on the UniProt website to quickly identify the subcellular location of each protein, so that you can filter out cytosolic, mitochondrial, or secreted proteins from your list of hits.

Problem 2
There is a little difference between the wild-type and phosphorylated PSM counts for hit proteins.

Potential solution
Ensure that kinase used to treat the CTD substrate is active.You can run an electrophoretic mobility shift assay (EMSA) to check for kinase activity or use matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry to determine the approximate number of phosphorylation groups that were added to the 26X CTD substrate.If fewer than 10 phosphate groups are added to the CTD substrate, this might not be enough to see differential protein recruitment in a pulldown experiment.

RESOURCE AVAILABILITY Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Yan Jessie Zhang (jzhang@cm.utexas.edu).

Technical contact
Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Haley A. Hardtke (hhardtke@utexas.edu).

Materials availability
There are no unique reagents associated with this protocol.

: 7 days 1 .
Culture cells for the pull-down experiment.Ensure that the cells have been in culture for a few passages and are fully equilibrated with media and culture conditions prior to starting the pulldown experiment.a.For HEK293 cells approximately 200 million cells are needed for one pull-down experiment, this is the equivalent of approximately 6 confluent T175 flasks.At minimum, 50 million cells per sample are needed.Thus, you can have a maximum of 4 samples per pull-down experiment.b.Seed 1 extra flask to count and estimate cell viability.2. Detach cells from the culture dish.a. Discard medium from the flask (or culture dish).b.Wash cells with sterile 13 PBS equilibrated to 20 C-22 C. c. Add Trypsin-EDTA to flask and place flask in incubator at 37 C for a maximum of 2 min.d.Use microscope to ensure cells have fully detached from the flask.e. Quench trypsin-EDTA with DMEM complete media.

f.
Pellet cells by centrifuging for 5 min at 200 g at 20 C-22 C. 3. Wash cells.a. Carefully resuspend cells in 1 mL of ice-cold sterile 13 PBS.b.Transfer cell suspension to a 2 mL tube.c.Pellet cells by centrifuging for 5 min at 200 g at 4 C. d.Determine packed cell volume (PCV)
h. Pellet cells by centrifugation at 5,500 g for 25 min at 4 C. Note: Store pellet at À20 C 16-18 h or flash freeze it for long-term storage at À80 C if immediate purification is not required.3. Lyse cells.a. Resuspend cells from both pellets in 50 mL CTD lysis buffer.b.Sonicate resuspended cells with five 30 s cycles.For each 30 s cycle, set the on time to 1 s, the off time to 5 s, and the amplitude to 90 A. Let the sample rest 3 min on ice between cycles.Note: Maintain the sample on ice throughout the sonication process to prevent overheating.c.Pellet cell lysate by centrifugation at 15,000 g for 35 min at 4 C. 4. Purify HGST-26X CTD with nickel affinity resin.a. Add 5 mL of Ni-NTA beads to a gravity column.b.Equilibrate Ni-NTA beads with 20 column volumes (CV) CTD lysis buffer.Note: Ni-NTA column purification must be done at 4 C. c. Run supernatant from step 3c over the equilibrated nickel beads.d.Collect the flowthrough and run the flowthrough over the nickel beads a second time.

6 .
Transform the His-DYRK1a (residues 127-485) pET-28a plasmid containing an N-terminal hexahistidine tag into E. coli BL21 (DE3) cells.a. Add 50 ng of the His-DYRK1a plasmid to a 50 mL aliquot of E. coli BL21 (DE3) cells.b.Incubate plasmid-cell solution on ice for 30 min.c.Place tube containing the plasmid-cell solution in a water bath set to 42 C for 60 s to heat shock.d.Place tube on ice for 5 min after heat shock.e. Transfer heat shocked cells to a microcentrifuge tube containing 800 mL SOC media.f.Place tube in incubator set to 37 C and 200 rpm and shake sample for 1 h to recover cells.g.Spin cells down at 3,000 g for 3 min at 20 C-22 C. h.Discard supernatant and resuspend cells in 50 mL of LB media.i. Plate cells on LB agar plates that contain 50 mg/mL kanamycin.j.Place LB/Kanamycin plates in a 37 C incubator for a minimum of 16 h.Colonies should be visible after 24 h.7. Express His-DYRK1a.a. Add 50 mL of 50 mg/mL kanamycin to Erlenmeyer flask that contains 50 mL of LB media, so that the final concentration of kanamycin in the LB media is 50 mg/mL.b.Inoculate the 50 mL LB/Kanamycin flask with cells from a colony on the plate from step 6j.c.Grow starter culture in a shaking incubator 16-18 h at 37 C with shaking at 200 rpm.d.Inoculate two 1 L terrific broth (TB) cultures with 10 mL starter culture and 50 mg/mL kanamycin.e. Grow 1 L cultures at 37 C with shaking at 200 rpm until the OD reaches 1.0.f.Induce cultures with IPTG so that the final concentration of IPTG in each 1 L culture is 0.5 mM and reduce the temperature of the shaker to 16 C to promote proper protein folding during expression.g.Let cultures incubate in the shaker 16-18 h at 16 C and 200 rpm.h.Pellet cells at by centrifugation 5,500 g for 25 min at 4 C. Note: Store pellet at À20 C 16-18 h or flash freeze it for long-term storage at À80 C if immediate kinase purification is not required.8. Lyse cells.

Figure 2 .
Figure 2. DYRK1a kinase purification (A) Size-exclusion chromatogram for DYRK1a kinase run on a Superdex 200 column (GE).(B) SDS-PAGE of pooled DYRK1a kinase after running size-exclusion chromatography.Sample was run on a 15% gel.

11 .
Transform the ERK2/MEK1 pET-28a plasmid into E. coli BL21 (DE3) cells.a. Add 50 ng of the ERK2/MEK1 plasmid to a 50 mL aliquot of E. coli BL21 (DE3) cells.b.Incubate plasmid-cell solution on ice for 30 min.c.Place tube containing the plasmid-cell solution in a water bath set to 42 C for 60 s to heat shock.d.Place tube on ice for 5 min after heat shock.
d. Inoculate two 1 L Luria-Bertani broth (LB) cultures with 10 mL starter culture and 50 mg/mL ampicillin.e. Grow 1 L culture at 37 C with shaking at 200 rpm until the OD reaches 0.6.f.Induce cultures with IPTG so that the final concentration of IPTG in each 1 L flask is 0.5 mM.g.After induction cultures were incubated at 37 C for 4 h with shaking at 200 rpm.h.Pellet cells by centrifugation at 5,500 g for 25 min at 4 C.Note: Store pellet at À20 C for 16-18 h or flash freeze it for long-term storage at À80 C if immediate kinase purification is not required.13.Lyse cells.a. Resuspend cell pellet in 50 mL ERK2 lysis buffer.b.Sonicate resuspended cells with five 30 s cycles.For each 30 s cycle, set the on time to 1 s, the off time to 5 s, and the amplitude to 90 A. Let the sample rest 3 min on ice between cycles.Note: Keep the sample in ice throughout the sonication process to prevent the sample from heating up during sonication.

Figure 3 .
Figure 3. SDS-PAGE of pooled ERK2 kinase after eluting the to sample from a Mono Q column Sample was run on a 15% gel.

Figure 4 .
Figure 4. MALDI-TOF spectra for an untreated CTD sample (black) and a DYRK1a-treated CTD (blue)The molecular weights for the highest peak is shown for each spectra.The difference between the mass of the kinase treated sample versus the untreated sample suggests that approximately 10 phosphate groups were added to the CTD in the treated sample.

Protocolc.
Pellet cell lysate by centrifugation at 15,000 g for 35 min at 4 C. 14. Purify ERK2 with nickel affinity resin.a. Equilibrate 5 mL of Ni-NTA beads with 20 CV ERK2 lysis buffer.Note: Ni-NTA column purification can be done at 20 C-22 C. b.Run supernatant from step 13c over the equilibrated nickel beads.c.Collect the flowthrough and run the flowthrough over the nickel beads a second time.d.Wash the column with 10 CV of ERK2 lysis buffer.e. Elute protein with 4 CV of ERK2 elution buffer.f.Run SDS-PAGE gel to confirm purity.15.Further purify ERK2 by anion exchange chromatography.a. Prepare a dialysis bag from snakeskin dialysis tubing (10,000 MWCO) and transfer the eluted protein into the membrane bag.b.Put dialysis bag in 1 L of ERK2 Mono Q buffer A and dialyze for 16-18 h at 4 C. c. Equilibrate Mono Q column (GE Life Sciences) with ERK2 Mono Q buffer A.

16 .
Treat CTD with kinase.a. Set up 120 mL kinase reactions as shown in the table below.Be sure to set up a control sample in parallel with the treated sample where 30 mL of ddH 2 O is added to the reaction mix in place of the kinase.Note: 0.4 mg/mL kinase was added to the kinase reaction (for final concentration of 0.1 mg/mL, regardless of the kinase used).STAR Protocols 5, 103277, September 20, 2024 Protocol Note: The final reaction volume should be 120 mL, where 100 mL will be used in the pull-down experiment and 20 mL will be saved to confirm phosphorylation by mass spectrometry and gel electrophoresis.Example MALDI-TOF mass spectra of untreated and kinase-treated samples are shown in Figure 4. b.Pipette up and down thoroughly to ensure the reaction solution is well mixed.c.Place reaction tubes into an Eppendorf thermomixer C. Set shaking to 600 rpm and the temperature to 30 C. d.The reaction time varies based on kinase activity.See table below for suggested duration of kinase reaction.17.Prepare GST beads.a. Pipette 200 mL of a slurry of 50% glutathione beads into a microcentrifuge tube.

Figure 5 .
Figure 5. Example volcano plot that compares hits from control versus kinase-treated sample Figure created with BioRender.com.
Prepare fresh every time and filter the buffer using a 0.45 mM filter after preparing.Add 2-mercaptoethanol directly before use.Keep ERK2 Mono Q buffer B at 4 C. Prepare fresh every time and filter the buffer using a 0.45 mM filter after preparing.Prior to the experiment, place an aliquot of buffer B on ice and supplement the aliquot with the necessary amount of protease and phosphatase inhibitors.Prior to the experiment, place an aliquot of high salt buffer on ice and supplement the aliquot with the necessary amount of protease and phosphatase inhibitors.
*Add 2-mercaptoethanol directly before use.ERK2 lysis buffer can be stored at 20 C-22 C indefinitely.Protect the buffer from light by wrapping it in tin foil to prevent degradation of imidazole.Aliquot the amount of ERK2 lysis buffer that is needed for individual experiments and supplement the aliquot with the necessary amount of 2-mercaptoethanol.*Add2-mercaptoethanoldirectlybeforeuse.ERK2 elution buffer can be stored at 20 C-22 C indefinitely.Protect the buffer from light by wrapping it in tin foil to prevent degradation of imidazole.Aliquot the amount of ERK2 elution buffer that is needed for individual experiments and supplement the aliquot with the necessary amount of 2-mercaptoethanol.Place the aliquot at 4 C to equilibrate the buffer to purification conditions.*Add2-mercaptoethanoldirectlybeforeuse.Keep ERK2 Mono Q buffer A at 4 C. Prepare fresh every time and filter the buffer using a 0.45 mM filter after preparing.Alternatives: To utilize multiple kinases in substrate treatment, adjust the final concentrations of Tris-HCl and MgCl 2 in the kinase buffer solution accordingly.For instance, if using two different kinases, prepare a 53 kinase buffer instead of the standard 43 concentration.ATP (8 mM)Dissolve 20.1 mg of ATP (507.18 g/mol) in 4 mL of ddH 2 O. Adjust the solution pH to 7.0 with 1 M Tris-HCl pH 7.5.Bring the final volume of the solution to 5 mL.Aliquot 1 mL of the 8 mM ATP stock into microcentrifuge tubes and store at À20 C for up to 2 years.Thaw ATP as needed.Avoid multiple freeze/thaw cycles.*(Continuedonnextpage)ProtocolElutionbuffer Supplement 1 mL high salt wash buffer with 20 mM reduced glutathione (stock 1000 mM).No additional protease and phosphatase inhibitors are necessary for this solution.Prepare the glutathione stock solution fresh every time.*Add2-mercaptoethanol directly before use.Buffer C can be stored at 20 C-22 C indefinitely.Prior to the experiment, place an aliquot of buffer C on ice and supplement the aliquot with the necessary amount of 2-mercaptoethanol.*Addprotease and phosphatase inhibitors (1003) directly before use.Low salt wash buffer can be stored at 20 C-22 C indefinitely.Prior to the experiment, place an aliquot of low salt buffer on ice and supplement the aliquot with the necessary amount of protease and phosphatase inhibitors.
-18 h at 4 C. Note: Use snap-cap tubes for this to ensure that your samples do not open overnight.
18. Pellet cells prepared in the before you begin section by spinning at 200 g for 5 min.Discard PBS supernatant.19.Extract cytoplasmic proteins.a. Resuspend washed cells in 1 PCV (approximately 1 mL) of buffer A. b.Vortex tubes and incubate on ice for 15 min.c.Centrifuge tubes at 15,000 g for 10 min at 4 C. d.Carefully remove the supernatant from each tube and save it as the cytoplasmic fraction.20.Extract nuclear proteins.a. Gently resuspend cell debris in 1 PCV (approximately 1 mL) of buffer A by pipetting up and down a few times.b.Spin down at 15,000 g for 1 min at 4 C. c. Discard the supernatant.d.Resuspend cell debris in 1 PCV of buffer B and supplement solution a 1:1000 dilution of benzonase to reduce protein:DNA interactions.i. Example: If the approximate volume of the solution is 1,200 mL add 1.2 mL of benzonase.e. Incubate the lysate/benzonase solution at 20 C-22 C for 1 h.f.Centrifuge tubes at 15,000 g for 10 min at 20 C-22 C. g.Collect the supernatant as the nuclear fraction.
22. Set up pull-down.a.Split nuclear protein extract evenly between samples.i.For example, if you have two samples, divide the nuclear protein extract equally by adding 500 mL extract to each tube with beads.Since 200 million cells were lysed, each sample should contain lysate from approximately 100 million cells.For optimal results, aim for 50 million cells per sample.In a standard CTD pull-down experiment, split the sample evenly among four tubes, comprising two control samples and two treated samples.b.After adding nuclear protein lysate to CTD-bound beads, incubate the tubes on a nutator for 24-48 h at 4 C.
Note:We typically incubate for 48 h.23.Wash pull-down samples and elute in preparation for MS/MS.a. Centrifuge samples at 4,000 g for 2 min at 4 C and carefully remove the supernatant.

Table 1 .
Example of output count files from the pull-down experiment 1aving a p value of 0.05 or lower.A volcano plot was generated by plotting the log2 fold change versus the -log10 of the calculated p value in GraphPad Prism (version 10.2.3).See supplementary table 1 in Moreno et al. for an example of expected results.1